Determinants of tumor sensitivity to EGFR-targeted antibodies This project has focused on understanding the critical structural determinants of anti-tumor antibodies that promote tumor-specific, antibody-dependent cellular cytotoxicity (ADCC), defining the antibody structural features and treatment strategies that maximize ADCC and promote the induction of adaptive immunity. Recently, our focus has shifted to analyzing the tumor-intrinsic factors that sensitize tumor cells to antibody therapy. We hypothesized that siRNA screening focused on genes functionally linked to the EGFR signaling pathway would identify tumor-intrinsic genes that regulate the tumor cell response to antigen engagement and ADCC promotion by monoclonal antibodies. To test this hypothesis, we have developed and applied a customized 638-element siRNA library containing genes known to functionally interact with EGFR (the EGFR functional interactome). Using this library, we identified a restricted number of genes whose knockdown selectively alters tumor cell viability in the presence of panitumumab and other EGFR inhibitors. We have identified clusters of genes known to act together in discrete sub-pathways, which we predict will be important for regulation of EGFR family-directed signaling inhibition and ADCC. This project will dissect immunologic and signaling mechanism contributions to antibody efficacy, and then identify efficacy-sensitizing genes for signaling and ADCC. Specific Aim 1 is to determine the roles of signaling inhibition and ADCC in mediating the efficacy of EGFR pathway-directed monoclonal antibodies. Specific Aim 2 is to define the elements of the EGFR interactome that modify target cell death in response to antibody engagement using cell culture-based functional tests to confirm the mechanisms by which siRNA depletion enhances tumor cell killing and in vivo models. In vitro and in vivo validation studies will identify new antibody-based therapy combinations. These studies will also identify those genes that regulate cellular sensitivity to EGFR inhibition and ADCC sensitivity. Specific Aim 3 is to determine the influence of mutations in critical signaling genes on EGFR antibody-targeted cytotoxicity. The completion of these aims will yield an improved understanding of the critical mechanisms that underlie successful antibody therapy, and will form the basis for future monoclonal antibody treatment strategies.